Related Experiment Video
Updated: Sep 4, 2026
Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques
Published on: March 13, 2015
Evaluating HbA1c measurement in haemoglobinopathy patients: Is transition beyond HPLC warranted for improved clinical
Rinini Dastidar1, Amit Halder1
1Department of Clinical Biochemistry, Nirnayan Health Care Pvt. Ltd., 145 Rajarhat Main Road, Kolkata 700059, India.
Diabetes mellitus is rapidly increasing in India, where coexistence of hemoglobinopathies such as HbE and β-thalassemia complicates accurate HbA1c estimation due to potential analytical interferences, particularly in cation-exchange high-performance liquid chromatography (CX-HPLC)-based assays. Capillary electrophoresis (CE) has been proposed as an alternative method with improved variant resolution in such populations. In this study, we evaluated the analytical comparability of CX-HPLC and CE for HbA1c measurement in hemoglobinopathy cases from Eastern India. A retrospective analysis was conducted on 3500 patient samples screened for hemoglobinopathies at Nirnayan Health Care Private Limited, Kolkata (January 2025). Hemoglobin variant analysis was performed using the Bio-Rad Variant II system, while HbA1c estimation was carried out using CX-HPLC (Bio-Rad Variant II Turbo; HbA1c mode ∼ 1.5 min) and CE (Sebia Minicap Flex Piercing; HbA1c mode ∼ 2.5 min). Statistical analyses, including Pearson correlation, Bland-Altman analysis and box-plot visualization, were performed using GraphPad Prism 9. Of the total samples, 327 showed hemoglobinopathies, predominantly HbE trait (n = 157) and β-thalassemia carriers (n = 129), along with other variants. HbA1c values measured by CX-HPLC and CE exhibited excellent correlation (r = 0.9799, p < 0.0001) with minimal bias and clinically acceptable limits of agreement. Subgroup analyses showed strong agreement in HbE carriers (r = 0.9745) and β-thalassemia carriers (r = 0.9086) with comparable glycaemic classification across non-diabetic, pre-diabetic, and diabetic ranges, although minor classification shifts were observed near diagnostic thresholds in a small subset of cases. HbA1c measurement was unreliable in homozygous hemoglobinopathies due to the absence or marked reduction of HbA, irrespective of the analytical method. These findings demonstrate that modern CX-HPLC and CE provide analytically comparable HbA1c results even in the presence of common hemoglobin variants. Both methods may be used interchangeably for HbA1c testing in variant-rich Indian populations.
Diabetes mellitus is rapidly increasing in India, where coexistence of hemoglobinopathies such as HbE and β-thalassemia complicates accurate HbA1c estimation due to potential analytical interferences, particularly in cation-exchange high-performance liquid chromatography (CX-HPLC)-based assays. Capillary electrophoresis (CE) has been proposed as an alternative method with improved variant resolution in such populations. In this study, we evaluated the analytical comparability of CX-HPLC and CE for HbA1c measurement in hemoglobinopathy cases from Eastern India. A retrospective analysis was conducted on 3500 patient samples screened for hemoglobinopathies at Nirnayan Health Care Private Limited, Kolkata (January 2025). Hemoglobin variant analysis was performed using the Bio-Rad Variant II system, while HbA1c estimation was carried out using CX-HPLC (Bio-Rad Variant II Turbo; HbA1c mode ∼ 1.5 min) and CE (Sebia Minicap Flex Piercing; HbA1c mode ∼ 2.5 min). Statistical analyses, including Pearson correlation, Bland-Altman analysis and box-plot visualization, were performed using GraphPad Prism 9. Of the total samples, 327 showed hemoglobinopathies, predominantly HbE trait (n = 157) and β-thalassemia carriers (n = 129), along with other variants. HbA1c values measured by CX-HPLC and CE exhibited excellent correlation (r = 0.9799, p < 0.0001) with minimal bias and clinically acceptable limits of agreement. Subgroup analyses showed strong agreement in HbE carriers (r = 0.9745) and β-thalassemia carriers (r = 0.9086) with comparable glycaemic classification across non-diabetic, pre-diabetic, and diabetic ranges, although minor classification shifts were observed near diagnostic thresholds in a small subset of cases. HbA1c measurement was unreliable in homozygous hemoglobinopathies due to the absence or marked reduction of HbA, irrespective of the analytical method. These findings demonstrate that modern CX-HPLC and CE provide analytically comparable HbA1c results even in the presence of common hemoglobin variants. Both methods may be used interchangeably for HbA1c testing in variant-rich Indian populations.
